PROJECT SUMMARY Vascular endothelial dysfunction (VED) is a key feature of vascular aging underlying the predisposition of old adults to CVD. Sustained oxidative stress is a major driving force for VED by synergically dysregulating the production of vasodilator NO and vasoconstrictor endothelin-1(ET-1). But the underlying redox mechanisms re- main unclear. Novel approaches to reversing oxidative stress are particularly compelling given the ineffective- ness of general antioxidant therapies that targets extremely short-lived ROS. Protein S-glutathionylation (PrS- SG), a stable but reversible oxidant-induced posttranslational modification dominates the molecular mechanisms for redox signaling. Reversal of PrS-SG is catalyzed specifically by glutaredoxin which thereby is critical in redox regulation of cellular function. Dysfunction of glutaredoxin-1 (Glrx-1), a major actor in the de-glutathionylation is implicated in cardiac, and brain aging. Its role in vascular aging is unknown. This application is built upon a strong premise. Our published and preliminary studies indicate that Glrx1 deficiency and PrS-SG induction promote endothelial dysfunction and aging-associated metabolic syndromes and that during aging, aortic Glrx1 is decreased with a concomitant increase in endothelial PrS-SG. Evidence in the literature also supports a causal role of Pr-SSG in VED in that S-glutathionylation can uncouple eNOS and activate p21Ras, which are known to impair NO bioavailability and stimulate Erk1/2 dependent-ET-1 expression. We thus hypothesize that age-related Glrx1 downregulation promotes VED by dysregulating the integrated redox signaling of eNOS and ET-1, which can be reversed by replenishing Grlx1 in endothelium. This central hypothesis is tested by pursuing two specific aims: 1) To understand the relationship between Glrx1/PrS-SG and the onset and progression of VED with aging. Using C57BL6J mice, a well-characterized model of aging we will measure the temporal changes in aortic Glrx1, PrS-SG, and endothelium-dependent vasorelaxation in young, middle-aged and old mice, and longitudinal changes of flow-mediated dilation of femoral artery in vivo using a non-invasive Optical-Coherence-Tomography technique. To test the causal role of Glrx1 downregulation in VED, we will test whether VED is aggravated in Glrx1 knockout mice; 2) To test a new concept that replenishing Glrx1 in endothelium can reverse VED in aging. Using a novel inducible endothelial specific Glrx1 transgenic mouse model, we will determine the impact on VED and the integrated signaling of eNOS and ET-1 of Glrx1 transgene expression in young, middle-aged, and old mice. The positive results in the R21 grant will help advance our understanding how redox signaling mediates the multifaceted effects of aging on ECs, promoting VED, and offer a new antioxidant therapeutic strategy to restore vascular function in older adults.